The present invention relates generally to the field of neuroprotectants and more specifically to the use of inhibitors of calcium-stimulated proteases, such as calpain, as therapeutics for neurodegeneration.
Neural tissues, including brain, are known to possess a large variety of proteases, including at least two calcium-stimulated proteases, termed calpain I and calpain II, which are activated by micromolar and millimolar Ca.sup.2+ o concentrations, respectively. Calpains are a family of calcium activated thiol proteases that are present in many tissues. Calpain II is the predominant form, but calpain I is found at synapses and is thought to be the form involved in long term potentiation and synaptic plasticity.
Thiol proteases are distinguished from serine proteases, metalloproteases and other proteases by their mechanism of action and by the amino acid residue (cysteine) that participates in substrate attack. Although several thiol proteases are produced by plants, these proteases are not common in mammals, with cathepsin B (a lysosomal enzyme), other cathepsins and the calpains being among the few representatives of this family that have been described in mammals. Calpain I and calpain II are the best described of these, but several other members of the calpain family have been reported.
Other Ca.sup.2+ -activated thiol proteases may exist, such as those reported by Yoshihara et al. in J. Biol. Chem. 265:5809-5815 (1990). The term "Calpain" is used hereinafter to refer to any Ca.sup.2+ -activated thiol proteases including the Yoshihara enzyme and calpains I and II.
While Calpains degrade a wide variety of protein substrates, cytoskeletal proteins seem to be particularly susceptible to attack. In at least some cases, the products of the proteolytic digestion of these proteins by Calpain are distinctive and persistent over time. Since cytoskeletal proteins are major components of certain types of cells, this provides a simple method of detecting Calpain activity in cells and tissues. Specifically, the accumulation of the breakdown products ("BDP's") of spectrin, a cytoskeletal protein, has been associated with the activation of Calpain. In neural tissues, activation of Calpains, as evidenced by accumulation of these BDP's, has been observed in many neurodegenerative conditions, including denervation resulting from focal electrolytic lesions, genetic abnormalities, excitotoxicity, Alzheimer's disease, following ischemia in gerbils and following administration of the toxins kainate and colchicine in rats, when administered peripherally or centrally.
Commercially available in vitro inhibitors of Calpain include peptide aldehydes such as leupeptin (Ac-Leu-Leu-Arg-H), as well as epoxysuccinates such as E-64. These compounds are not useful in inhibiting Calpain in Central Nervous System ("CNS") tissue in vivo because they are poorly membrane permeant and, accordingly, do not cross the blood brain barrier very well. Also, many of these inhibitors are poorly specific and will inhibit a wide variety of proteases in addition to Calpain. These commercially available compounds are based upon peptide structures that are believed to interact with the substrate binding site of Calpain. Active groups associated with the Calpain inhibitors then either block or attack the catalytic moiety of Calpain in order to inhibit the enzyme.
In addition, other types of compounds thought to possess in vitro Calpain inhibitory activity that are not commercially available have been reported. Examples of such compounds include the peptide diazomethanes. See Rich, D. H., Inhibitors of cysteine proteinases, in Protease Inhibitors, A. J. Barrett and G. Salversen, Eds., Elsevier, New York, 1986, pp153-178, the disclosure of which is hereby incorporated by reference. These peptide diazomethanes are similarly thought to be poorly membrane permeant and non-specific.
There is some evidence that certain particular inhibitors of Calpain have certain therapeutic utilities. For example, leupeptin can facilitate nerve repair in primates. Loxastatin (also known as EST, Ep-460 or E-64d), a derivative of E-64, is believed to have utility in the treatment of muscular dystrophy. E-64d, while not having significant protease inhibitory activity itself, is believed to be converted to more potent forms, such as to E-64c, inside a mammalian body.
Evidence from electrophysiological studies suggests that one of the earliest factors in the chain of reactions leading to cell death is an increase in intracellular-free calcium as a consequence of Ca.sup.2+ channel opening and/or energy depletion. Intracellular calcium is likely to produce a large number of consequences, including the activation of a large number of enzymes, including proteases, such as Calpain, lipases and kinases. An increase in intracellular calcium is also thought to induce changes in gene expression.
Ischemia, head trauma and stroke have all been associated with the release of glutamate in amounts large enough to lead to excitotoxicity, the toxicity resulting from the actions of certain amino acids on neurons of the CNS. The excess glutamate and other factors, such as free radical damage of membranes or energy depletion, cause an increase in intracellular Ca.sup.2+. It is known that an excess of intracellular Ca.sup.2+ leads to several effects believed to be associated with neuronal cell damage, including destruction of cell structures through activation of phospholipase and Calpain, as well as free radical production resulting from activation of phospholipase and xanthine oxidase. Many other factors have been associated with neurotoxicity. For example, reductions in action potentials and changes in a wide variety of chemical markers are known to be associated with neurons exposed to ischemic conditions.
Notwithstanding the foregoing understanding of certain aspects of neurotoxicity, no effective therapy has yet been developed for most neurodegenerative diseases and conditions of the CNS. Millions of individuals suffer from these diseases and conditions. Thus, there is a need for therapies effective in treating and preventing these diseases and conditions.